Method and device for collecting and stabilizing a biological sample

ABSTRACT

A collection container and method for collecting a predetermined volume of a biological sample, and particularly a whole blood sample, includes an effective amount of at least one stabilizing agent. The stabilizing agent is able to stabilize nucleic acids in the biological sample at the point of collection to prevent enzymatic degradation of the nucleic acids. The stabilizing agents include cationic compounds, detergents, particularly cationic detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, and mixtures thereof.

FIELD OF THE INVENTION

[0001] The present invention is directed to a method and device forcollecting a biological sample, and particularly a whole blood sample,directly from a patient. More particularly, the invention relates toevacuated fluid sample containers having a stabilizing additivecontained therein for stabilizing nucleic acids immediately oncollection of a biological sample.

BACKGROUND OF THE INVENTION

[0002] Sample collection containers have been in common use for manyyears for collecting and storing blood and other body fluids or samples.Typically, the collection containers are glass or plastic tubes having aresilient stopper. These glass or plastic tubes are often used for bloodcollection.

[0003] Blood collection tubes are available where the tube is evacuatedto draw a volume of blood into the tube. The tubes can have variousadditives, such as ethylenediaminetetraacetic acid (EDTA) containedtherein for preparing the blood sample for a particular test. A commonadditive is an anticoagulation agent. Typically, the anticoagulationadditive is a buffered citrate or heparin in an aqueous solution. Theaqueous citrate is combined with the blood sample in a specified amountto determine the amount of an anticoagulant needed for conductingcertain tests. These devices can be used only for serological testingsince the additives do not stabilize the nucleic acids in the sample.During shipment, labile RNA molecules are degraded enzymatically so thatsubsequent RNA separation and analysis is difficult. Furthermore,mechanical irritation or disruption of cells during blood collection andtransport causes the induction of gene transcription with theconcomitant over- or underproduction of certain mRNA species.

[0004] Common additives including anticoagulants to maintain the bloodsample in an anticoagulated state are used for performing variousprocessing steps. For example, anticoagulants are typically used inblood samples prior to centrifuging to separate the blood into celllayers. An example of this type of sample tube containing ananticoagulant is disclosed in U.S. Pat. No. 5,667,963 to Smith et al.

[0005] In recent years there has been an increase in interest in thefield of biological, medical and pharmacological science in the study ofgene activities and nucleic acids obtained from biological samples. Inparticular, ribonucleic acids can provide extensive information of thegenetic origin and functional activity of the cell. This information maybe used in clinical practice to diagnose infections, detect the presenceof cells expressing oncogenes, detect heredity disorders, monitor thestate of host defense mechanisms and to determine the HLA type or othermarker of identity.

[0006] A number of methods exist for isolating RNA which entailsdisruption of the cell and liberating RNA into solution. Other methodsexist for protecting RNA from enzymatic digestion by endogenous RNases.The RNA can then be separated from the DNA and protein, which issolubilized along with the RNA. These processes are usually performed instepwise fashion rather than for simultaneously lysing cells,solubilizing RNA and inhibiting RNases. Some methods for lysing cellsand inhibiting RNases are known that use chaotropic salts ofguanidinium.

[0007] A commonly used process for isolating RNA involves homogenizingcells in guanidinium isothiocyanate, followed by the sequential additionof sodium acetates and phenol, and chloroform/isoamyl alcohol. Aftercentrifugation, RNA is precipitated from the upper layer by the additionof alcohol. Other methods include the addition of hot phenol to a cellsuspension, followed by alcohol precipitation.

[0008] Anionic and cationic surfactants are used to lyse cells andliberate cytoplasmic RNA. An example of a method for lysing cells andsimultaneously precipitating RNA and DNA from solution is disclosed inU.S. Pat. No. 5,010,183 to Macfarlane. In this process, the RNA is madeinsoluble. A 2% solution of the surfactant benzyldimethyln-hexadecylammonium chloride together with 40% urea and other additivesare added to a cell suspension. The suspension is then centrifuged torecover a pellet of the insoluble materials. The pellet is resuspendedin ethanol and the RNA and DNA are precipitated by the addition of asalt.

[0009] A method for analyzing RNA isolated from blood uses amplificationmethods including polymerase chain reaction to detect sequences of RNAin minute amounts. One difficulty in analyzing RNA is the separation ofthe RNA from the protein and the DNA in the cell before the RNA isdegraded by nucleases. RNase and other nucleases are present in theblood in sufficient quantities to destroy unprotected RNA. Therefore, itis desirable to use a method of isolating RNA from cells in a manner toprevent hydrolysis of RNA by nucleases.

[0010] The blood collection methods currently in common use are able tocollect and retain the blood for analysis at a later time. Thecollection device can include an anticoagulant to prevent coagulationduring storage. However, the nucleases present in the blood hydrolyzesome RNA species during storage and transport while mechanicalirritation or disruption of cells during blood collection causesinduction of some RNA species. These preanalytical sample handlingfactors result in under- or overrepresentation of mRNA species andeventual degradation of total RNA as determined by molecular diagnostictest methods. In addition, gene induction can result in increased levelsof RNA in the sample, which can give false results. Accordingly, thereis a continuing need in the industry for an improved method andcollection device for blood and other biological samples that preservethe in vivo transcription profile for nucleic acid-based tests.

SUMMARY OF THE INVENTION

[0011] The present invention is directed to a method and device forcollecting a biological sample. More particularly, the invention isdirected to a collection container and to a method of collecting abiological sample with a stabilizing additive to stabilize the sampleand preserve the in vivo transcription profile.

[0012] Accordingly, a primary aspect of the invention is to provide amethod and device for collecting a biological sample, and particularlywhole blood, directly from a patient in the presence of a stabilizer tostabilize and preserve RNA and prevent gene induction in the sample. Thestabilizing additive is present in an effective amount to stabilize thenucleic acids, particularly RNA, and stop gene induction.

[0013] A further object of the invention is to provide a method anddevice for stabilizing nucleic acids in a biological sample and to lysecells, bacteria, viruses and reticulocytes.

[0014] Another object of the invention is to provide a collectioncontainer for receiving and collecting a biological sample where thecontainer is pre-filled with a measured quantity of a nucleic acidstabilizing agent.

[0015] A further object of the invention is to provide a method forstabilizing a biological sample, and particularly whole blood,immediately upon collection from the patient.

[0016] Still another object of the invention is to provide a method forpreventing induced transcription of RNA in a biological sampleimmediately on collection of the biological sample.

[0017] Another object of the invention is to provide an evacuatedcontainer containing an effective amount of a nucleic acid stabilizingagent, where the container has an internal pressure to draw apredetermined volume of a biological sample into the container.

[0018] A further object of the invention is to provide a bloodcollection container for collecting an amount of blood and mixing theblood with a nucleic acid stabilizing agent at the point of collectionto preserve the nucleic acids and prevent gene induction such thatanalysis can be conducted at a later time.

[0019] Another object of the invention is to provide a method ofstabilizing blood by collecting the blood sample in a container having anucleic acid stabilizing agent and a buffer. The nucleic acidstabilizing agent can be a detergent, a chaotropic salt, RNaseinhibitors, chelating agents, or mixtures thereof. The pH of theresulting mixture is adjusted to stabilize the nucleic acids and promoteefficient recovery of the analyte.

[0020] Still another object of the invention is to provide a method ofstabilizing nucleic acids in a blood sample collection device at aboutpH 2 to about pH 5 in the presence of at least one stabilizing agent.

[0021] The objects of the invention are basically attained by providingan apparatus for collecting a biological sample. The apparatus includesa container comprising a side wall, a bottom wall, and an open enddefining an internal chamber, and a closure closing the open end. Thecontainer includes at least one stabilizing component in an effectiveamount to stabilize and preserve the biological sample. The containercan be pre-filled with the stabilizing agent.

[0022] The objects of the invention are further attained by providing amethod of stabilizing a biological sample comprising the steps of:providing a sample collection container having a side wall, and a bottomdefining an internal chamber. The container contains at least onestabilizing agent in an amount and concentration sufficient to stabilizeand preserve a biological sample. A biological sample is obtained andimmediately introduced into the container and the biological sample ismixed with the stabilizing agent to form a stabilized biological sample.

[0023] The objects of the invention are also attained by providing amethod of collecting and stabilizing a whole blood or other biologicalsample. The method comprises providing a sample collection containerhaving a side wall, a bottom wall and a closure member forming aninternal chamber. The container encloses an effective amount of anaqueous solution or dispersion of a nucleic acid stabilizing agent tostabilize and preserve nucleic acids and/or the transcriptional profilein a whole blood sample. The internal chamber has pressure less thanatmospheric pressure. A whole blood sample is collected directly from apatient in the collection container and the blood sample is mixed withthe stabilizing agent to form a stable whole blood sample.

[0024] These objects, advantages and other salient features of theinvention will become apparent from the annexed drawing and thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0025] The following is a brief description of the drawing, in which:

[0026]FIG. 1 is a cross-sectional side view of the container in oneembodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is directed to a method and device forstabilizing and preserving a biological sample. More particularly, theinvention is directed to a method and device for stabilizing nucleicacids and/or preventing gene induction in a biological sample. Inpreferred embodiments of the invention, the device is a pre-filledcontainer containing an amount of a nucleic acid stabilizing agent.

[0028] The biological sample can be a body fluid withdrawn from apatient. In one embodiment, the biological fluid is whole blood.Examples of other biological samples include cell-containingcompositions such as red blood cell concentrates, platelet concentrates,leukocyte concentrates, plasma, serum, urine, bone marrow aspirates,cerebral spinal fluid, tissue, cells, and other body fluids.

[0029] In a preferred embodiment, the device 10 is for drawing a bloodsample directly from a patient for stabilizing the nucleic acidsimmediately at the point of collection. Referring to FIG. 1, device 10includes a container 12 defining a chamber 14. In the embodimentillustrated, container 12 is a hollow tube having a side wall 16, aclosed bottom end 18 and an open top end 20. Container 12 is dimensionedfor collecting a suitable volume of a biological fluid. A resilientclosure 22 is positioned in open top end 20 to close container 12.Preferably, closure 22 forms a seal capable of effectively closingcontainer 12 and retaining a biological sample in chamber 14. Aprotective shield 23 overlies closure 22.

[0030] Container 12 can be made of glass, plastic or other suitablematerials. Plastic materials can be oxygen impermeable materials orcontain an oxygen impermeable layer. Alternatively, container 12 can bemade of a water and air permeable plastic material. Preferably, chamber14 maintains a pressure differential between atmospheric pressure and isat a pressure less than atmospheric pressure. The pressure in chamber 14is selected to draw a predetermined volume of a biological sample intochamber 14. Typically, a biological sample is drawn into chamber, 14 bypiercing closure 22 with a needle 24 or cannula as known in the art. Anexample of a suitable container 12 and closure 22 are disclosed in U.S.Pat. No. 5,860,937 to Cohen, which is hereby incorporated by referencein its entirety.

[0031] Container 12 is preferably made of a transparent material.Examples of suitable transparent thermoplastic materials includepolycarbonates, polyethylene, polypropylene, polyethylene-terephthalate. Container 12 has a suitable dimension selected according to therequired volume of the biological sample being collected. In oneembodiment, container 12 has a tubular shape with an axial length ofabout 100-mm and a diameter of about 13-mm to 16-mm.

[0032] Closure 22 is made of a resilient material capable of maintainingan internal pressure differential less than atmospheric and that can bepierced by a needle to introduce a biological sample into container 12.Suitable materials for closure include, for example, silicone rubber,natural rubber, styrene butadiene rubber, ethylene-propylene copolymersand polychloroprene.

[0033] Container 12 also contains a stabilizing additive 26. Thestabilizing additive 26 is preferably a liquid containing a stabilizingagent and is included in an effective amount to mix with the biologicalsample and stabilize the nucleic acids and/or prevent gene induction ofthe cells or nucleic acids contained therein. In one embodiment, theinternal pressure of container 12 and the volume of stabilizing additive26 are selected to provide the necessary concentration of thestabilizing agent for the volume of the biological sample collected. Inone preferred embodiment, the internal pressure of container 12 isselected to draw a predetermined volume of about 2.5 ml of a biologicalsample into container 12 containing an effective volume of stabilizingadditive 26 for stabilizing the volume of the biological sample. Inalternative embodiments, container 12 can have an internal pressure atsubstantially atmospheric pressure.

[0034] In one embodiment, container 12 is made of a plastic that iswater and gas permeable. Water loss by evaporation of the stabilizingagent through the permeable wall of the container increases theconcentration of the stabilizing agent and decreases the pressure withinthe container. The diffusion of oxygen through the wall of the tube hasthe effect of decreasing the vacuum in the container. The water andoxygen permeability properties of the container are selected to maintainthe desired pressure differential within the container for the desiredshelf life of the container. The shelf life is optimized by balancingthe oxygen permeability with the water loss. Preferably, the containerhas a shelf life of about one year.

[0035] Stabilizing additive 26 is typically an aqueous solution ordispersion of at least one active stabilizing agent that is included inthe container as a pre-filled container. Stabilizing additive 26preferably contains at least one stabilizing agent in a concentrationcapable of stabilizing nucleic acids in the biological sample, andparticularly a whole blood sample. The stabilizing agents are preferablyable to stabilize effectively DNA and RNA including mRNA, tRNA and cRNA.Examples of suitable stabilizing agents for stabilizing and preservingnucleic acids and/or preventing gene induction include cationiccompounds, detergents, chaotropic salts, ribonuclease inhibitors,chelating agents, and mixtures thereof. A suitable ribonucleaseinhibitor is placental RNAse inhibitor protein. Examples of chaotropicsalts include urea, formaldehyde, guanidinium isothiocyanate,guanidinium hydrochloride, formamide, dimethylsulfoxide, ethylene glycoland tetrafluoroacetate.

[0036] The stabilizing agent can also include another component fortreating the biological sample. For example, chemical agents can beincluded to permeabilize or lyse viruses and cells. Other componentsinclude proteinases, phenol, phenol/chloroform mixtures, alcohols,aldehydes, ketones and organic acids.

[0037] The detergents can be anionic detergents, cationic detergents ornonionic detergents. The anionic detergent can be, for example, sodiumdodecyl sulfate. Nonionic detergents can be, for example, ethylene oxidecondensation products, such as ethoxylated fatty acid esters ofpolyhydric alcohols. A preferred nonionic detergent is a polyoxyethylenesorbitan monolaurate sold under the trade name TWEEN 20 by SigmaChemical Co. The detergents are included in an effective amount to lysethe cells and form micelles and other complexes with the nucleic acids.

[0038] In preferred embodiments, the stabilizing agent is a cationiccompound having the general formula YR₁R₂R₃R₄ X wherein Y is nitrogen orphosphorous; R₁, R₂, R₃, and R₄ are independently branched ornon-branched alkyl, C₆-C₂₀ aryl, or C₆-C₂₆ aralkyl, and X is an organicor inorganic anion.

[0039] The anion can be an anion of an inorganic acid such as the HXwhere X is fluorine, chlorine, bromine or iodine, with chlorine andbromine being preferred. The anion can also be the anion of a mono-, di-or tricarboxylic acid. Typically, the anion of the cationic compound isselected from the group consisting of phosphate, oxalate, malonate,succinate, citrate, bromide and chloride.

[0040] When R₁, R₂, R₃, and R₄ are aryl groups, the aryl groupsindependently can be, for example, phenyl, lower alkyl-substitutedbenzyl, and/or halogenated benzyl. In one embodiment R₁ is a C₁₂, C₁₄,or C₁₆ alkyl and R₂, R₃, and R₄ are methyl groups. In a preferredembodiment, Y is nitrogen and the stabilizing agent is a quaternaryamine. Suitable quaternary amines include alkyltrimethylammonium wherethe alkyl group has 12, 14 or 16 carbons. One preferred cationiccompound is tetradecyltrimethyl ammonium oxalate. Other suitablequaternary amines include alkyltrimethylammonium where the alkyl groupincludes 12, 14, 16 or 18 carbons. Examples of suitable quaternary aminesurfactants are disclosed in U.S. Pat. No. 5,728,822 to Macfarlane,which is hereby incorporated by reference in its entirety.

[0041] In preferred embodiments of the invention the stabilizing agentis a cationic compound and includes a proton donor. It has been foundthat the addition of a proton donor to the cationic compounds increasesthe ability of the cationic compounds to stabilize the nucleic acids inthe biological sample. Examples of suitable proton donors includemonocarboxylic acids, alkenyl carboxylic acids, C₂-C₆ aliphatic mono-and dicarboxylic acids, aliphatic ketodicarboxylic acids, amino acids,mineral acids and mixtures thereof. Examples of suitable aliphaticcarboxylic acids include C₁-C₆ alkyl carboxylic acids, such as aceticacid, propionic acid, n-butanoic acid, n-pentanoic acid, isopentanoicacid, 2-methylbutanoic acid, 2,2 dimethylpropionic acid, n-hexanoicacid, n-octanoic acid, n-decanoic acid, and dodecanoic acid. Examples ofalkenyl carboxylic acids include acrylic acid, methacrylic acid,butenoic acid, isobutenoic acid and mixtures thereof.

[0042] The dicarboxylic acids of the proton donor are selected from theconsisting of oxalic acid, malonic acid, succinic acid, glutaric acid,adipic acid and mixtures thereof. Examples of hydroxyl-containing acidsinclude tartaric acid and malic acid. Suitable amino acids are selectedfrom the group consisting of glycine, alanine, valine, leucine,iso-leucine and mixtures thereof.

[0043] The quantity of stabilizing additive 26 in container 12 isdetermined by the internal volume of container 12, the internal pressureand the volume of the biological sample drawn into the container. In theillustrated embodiment, container 12 has an axial length of about 100-mmand a diameter of about 16-mm and has an internal pressure to draw abiological sample of about 2.5-ml. Stabilizing additive 26 typicallycontains about 50 mg to about 90 mg per ml of the carrier liquid.Preferably, stabilizing additive 26 is an aqueous medium containingabout 60 mg/ml to about 80 mg/ml, and most preferably about 70 mg/ml.The volume of stabilizing additive 26 in container 12 is about 6 to 8ml, and preferably about 7 ml.

[0044] In one preferred embodiment, stabilizing additive 26 includesabout 70 mg/ml of a nucleic acid stabilizing agent and is mixed withwhole blood drawn directly from a patient. The blood is mixed with theliquid in a ratio of about 1:2 to about 1:3.5, preferably about 1:2.5 toabout 1:1.3, and most preferably about 1:2.7 to about 1:2.8 by volume.

[0045] The concentration of the stabilizing agent is sufficient tostabilize the nucleic acids. In one preferred embodiment, the biologicalsample is whole blood. The concentration of the stabilizing agent in anamount of about 8 to about 12 mg/ml of blood, preferably about 9 toabout 11 mg/ml, and more preferably about 10 mg/ml of blood.

[0046] The method of the invention is performed by obtaining abiological sample and introducing the sample into the containercontaining the stabilizing additive. In preferred embodiments thebiological sample is prepared and immediately introduced directly intothe collection container. In certain embodiments, the biological sampleis introduced directly into the collection container with out anyintervening process steps. It has been found that collecting thebiological sample directly from the patient, such as when collecting awhole blood sample, and introducing the sample directly into thecontainer substantially prevents or reduces the decomposition of thenucleic acids that otherwise occur when the sample is stored beforecombining with the stabilizing agent. In addition, is has been foundthat the introduction of the biological sample immediately uponcollection or preparation reduces or prevents gene induction.

[0047] The cationic compounds are preferred stabilizing agents since thecationic group is able to effectively attack the negatively chargedgroups of the nucleic acids. The cationic groups of the compound fornon-covalent bonds with the negatively charged phosphate groups of thenucleic acid backbone. The hydrophilic, and particularly the aliphatic,portion of the detergent molecules form precipitatable micelles andcomplexes containing the nucleic acids. The resulting micelles isolatethe nucleic acids from the endogenous ribonucleases to prevent enzymaticdegradation. The cationic compounds produce a stabilized whole bloodsample that can be transported at ambient temperature to a laboratorywhere the nucleic acids can be isolated from the sample.

[0048] It has been found that the recovery and stabilization of nucleicacids in the biological sample is dependent on the pH of the biologicalsample and stabilizing agent. The pH of the resulting mixture can rangefrom about pH 2 to about pH 12, preferably about pH 2 to about pH 10,and more preferably about pH 3 to about pH 8. The life of the nucleicacids in this range will vary depending on the biological sample, theratio of the amount of the biological sample to the amount of thestabilizing agent, and the particular stabilizing agent used. The shelflife of the stabilized nucleic acids in this pH can range from about 24hours to several days.

[0049] The pH of the resulting mixture will vary depending on thebiological sample being stabilized. In one embodiment of the invention,the biological sample is whole blood and the mixture of the whole bloodand the stabilizing agent is adjust to about pH 2 to about pH 5. Nucleicacids stabilized with cationic compounds adjusted to about pH 2 to aboutpH 5 are stable at ambient temperature for several days. It has beenfound that optimum long term stabilization of nucleic acids in wholeblood is obtained at about pH 3.6 to about pH 3.8. In other biologicalsamples, the pH is adjusted to stabilize the mixture. For example, ithas been found that eucaryotic cell cultures and bacteria are stabilizedat pH 4 to about pH 8, and preferably at about pH 6 to about pH 8.

[0050] The pH of the mixture of the biological sample and stabilizingagent can be adjusted by the addition of a suitable buffer. An exampleof a buffer that has been found to be effective in adjusting the pH ofthe biological sample is tartaric acid. Other buffers and pH adjustingagents as known in the art can also be used. The pH of the buffer can beadjusted to the desired range by the addition: of sodium hydroxide.

[0051] The nucleic acids, either DNA or RNA can be separated from thestabilized biological sample using various processes as known in theart. It has been found that the stabilizing agents can be separated fromthe nucleic acids during the purification protocol performed in thelaboratory to yield the purified nucleic acid.

[0052] Cationic compounds cause lysis of the cells and virus in thesample and precipitation of the nucleic acids in a complex with thecompound. The precipitated nucleic acids can be extracted from thecomplex by a phenol extraction or by a formamide buffer as known in theart. In a further embodiment, the detergent can be solubilized todisassociate the complex and leave the insoluble nucleic acids. Thecompound can be solubilized by treating the complex with a concentratedsolution of lithium chloride. Other methods of isolating and purifyingnucleic acids are disclosed in U.S. Pat. No. 5,990,301 to Colpan et al.,which is hereby incorporated by reference in its entirety.

EXAMPLE 1 Stabilization of RNA in Human Blood

[0053] This example demonstrates the effects of the ratio of the bloodto stabilizing agent and the concentration of the stabilizing agent.

[0054] Twenty-four samples were prepared for this comparison. Eachsample was prepared from 2.5 ml blood, drawn with a sodium citratecontaining blood collection device, and mixed with 7.5 ml of astabilization buffer containing 3% (w/v) tetradecyltrimethylammoniumoxalate and 125 mM and 200 mM tartaric acid, respectively, in a 12 mlpolyethylene tube. The pH of the buffer was adjusted with sodiumhydroxide to 3.3, 3.5 and 3.7, respectively. Samples were stored at roomtemperature for 25 hours and 72 hours, respectively. In order to isolatethe cellular RNA, the tubes were centrifuged at 5000×g for 10 minutes.The supernatant was discarded and the pellet was washed once with water.After additional centrifugation at 5000×g for 10 minutes, the pellet wasdissolved in 300 μl of a lysis buffer, i.e., buffer RLT (QIAGEN GmbH),diluted with 360 μl water and 40 μl proteinase K were added. After aproteinase digestion for 10 minutes at 55° C. the sample was centrifugedat 20,000×g for 3 minutes, the supernatant was transferred into a newtube and 350 μl of 98% ethanol were added. The sample was then appliedto a silica membrane containing spin column via centrifugation at 8000×gfor 1 minute. The spin column was washed once with a GITC containingwashing buffer-like buffer RW1 (QIAGEN GmbH) and two times with aethanol containing buffer-like buffer RPE (QIAGEN GmbH). The RNA wasthen eluted from the silica membrane with 2×40 μl of RNase free water.All samples were processed in duplicates.

[0055] The yield of the isolated RNA was determined by measuring theoptical density at 260 nm wavelength in a spectrophotometer andcalculating that 1 OD260 corresponds to a concentration of 40 μg RNA/ml.The integrity of the isolated RNA was proved by electrophoresis of 30 μlof the eluate in a denaturating agarose/formaldehyde gel, stained withethidium bromide. The yield of the RNA is presented in Table 1 and Table2. TABLE 1 125 mM Tartaric Acid Sample pH Storage Time (hours) Yield(μg) 1 3.3 24 8.4 2 3.3 24 7.6 3 3.5 24 9.5 4 3.5 24 9.8 5 3.7 24 13.3 63.7 24 17.2 7 3.3 72 7.2 8 3.3 72 6.8 9 3.5 72 10.3 10 3.5 72 10.9 113.7 72 14.8 12 3.7 72 16.1

[0056] TABLE 2 200 mM Tartaric Acid Sample pH Storage Time (hours) Yield(μg) 13 3.3 24 5.9 14 3.3 24 7.4 15 3.5 24 10.6 16 3.5 24 10.9 17 3.7 2417.2 18 3.7 24 18.5 19 3.3 72 5.1 20 3.3 72 5.3 21 3.5 72 7.2 22 3.5 727.1 23 3.7 72 13.3 24 3.7 72 16.6

[0057] the results show that for the blood volume of 2.5 ml mixed with7.5 ml of stabilization buffer containing 3% (w/v)tetradecyltrimethylammonium oxalate and 125 mM or 200 mM tartaric acid,respectively, the pH of 3.7 is optimal for the yield and integrity ofthe total RNA. With all pH values, the stabilization of the RNA, judgedby the integrity of the ribosomal RNA, was very good, but the yield ofthe isolated RNA was lower with the buffers adjusted to pH 3.3 and 3.5,respectively, than with the buffer adjusted to pH 3.7. However, even thelower yields achieved with the stabilization buffer adjusted to pH 3.3were comparable or slightly better than the yields achieved with acontrol method, the RNA isolation with the QIAamp® RNA Blood Mini Kit(QIAGEN Cat. No. 52303), which showed an average yield of 6.8 μg RNA per2.5 ml of blood.

EXAMPLE 2 Northern-Blot Anaylsis

[0058] This example shows the results of a Northern-Blot analysisperformed with blood samples from three different donors stored at roomtemperature for 1 hour, 24 hours, 48 hours and 72 hours.

[0059] 2.5 ml blood samples, drawn with a sodium citrate containingblood collection device, were mixed with 6.9 ml of stabilization buffercontaining 4% (w/v) tetradecyltrimethylammonium oxalate and 200 mMtartaric acid in a 16×100 mm polyethylene tube. Samples were stored atroom temperature for 1 hour, 24 hours 48 hours and 72 hours,respectively. In order to isolate the cellular RNA, the tubes werecentrifuged at 5000×g for 10 minutes. The supernatant was discarded andthe pellet was washed once with water. After additional centrifugationat 5000×g for 10 minutes, the pellet was dissolved in 300 μl of a lysisbuffer, i.e., buffer RLT (QIAGEN GmbH), diluted with 360 μl water and 40μl proteinase K were added. After a proteinase digestion for 10 minutesat 55° C. the sample was centrifuged at 20,000×g for 3 minutes, thesupernatant was transferred into a new tube and 350 μl of 98% ethanolwere added.

[0060] The sample was then applied to a silica membrane containing spincolumn by centrifugation at 8000×g for 1 minute. The spin column waswashed once with a GITC containing washing buffer-like buffer RW1(QIAGEN GmbH) and two times with a ethanol containing buffer-like bufferRPE (QIAGEN GmbH). The RNA was then eluted from the silica membrane with2×40 μl of RNase free water. A single sample was prepared for eachvariable. 2.5 μg of the isolated RNA were loaded onto a denaturatingagarose/formaldehyde gel, and after the electrophoresis the RNA wastransferred onto a nylon membrane. The nylon membrane was hybridizedsubsequently with a radioactive labeled RNA probe, which contained thesequence of an IFN-gamma inducible gene (GeneBank Acc.No. L07633)overnight at 60° C., washed several times at 60° C. with washing bufferscontaining 2×SSC/0.1% SDS to 0.5×SSC/0.1% SDS. The nylon membrane wasexposed subsequently to an X-ray film. As a control, RNA from the samedonor was isolated using TRIzol™ LS reagent (Life Technologies) directlyafter the blood draw and analyzed as described above.

[0061] The results show that the transcript levels of the IFN-gammainducible gene, which was used as a probe to hybridize the isolated RNA,was preserved over the entire time period with no visible change in theexpression level. The transcript levels were equal to the TRIzol™LScontrols. These controls represent the in vivo conditions of the sampleat the time point of the blood draw because the TRIzol reagent containsphenol combined with guanidine isothiocyanate and is considered as areagent that destroys cells immediately, denatures proteins andtherefore completely inhibits any biological activity. The comparison ofthe signal intensities from the stored samples with the TRIzol controlsin the Northern-Blot analysis indicates that the transcript levels ofthe IFN-gamma inducible gene were “frozen” immediately after addition ofthe stabilization buffer to the blood sample and did not change any moreduring storage.

EXAMPLE 3 Comparison of Blood Collection Device with Conventional EDTATube

[0062] This example compares the stabilization of RNA with thecollection device of the present invention and conventional EDTAcontaining tube.

[0063] 2.5 ml blood, drawn from one donor with a blood collectiondevice, containing 6.9 ml of stabilization buffer (4% (w/v)tetradecyltrimethylammonium oxalate, 200 mM tartaric acid, pH 3.7) in a16×100 mm polyethylene tube closed with a HEMOGARD™ closure (Becton,Dickinson and Company) and evacuated to a defined vacuum that drawed 2.5ml of blood when connected to the vein of the donor. Samples were storedat room temperature for 1 hour, 1 day, 3 days, 7 days and 10 days,respectively. In order to isolate the cellular RNA, the tubes werecentrifuged at 5000×g for 10 min. The supernatant was discarded and thepellet was washed once with water. After additional centrifugation at5000×g for 10 min, the pellet was dissolved in 360 μl of a resuspensionbuffer containing ammonium acetate and then 300 μl of a lysis buffer,i.e., buffer RLT (QIAGEN GmbH), and 40 μl proteinase K were added. Aftera proteinase digestion for 10 minutes at 55° C., the sample wascentrifuged at 20,000×g for 3 minutes, the supernatant was transferredinto a new tube and 350 μl of 98% ethanol were added. The sample wasthen applied to a silica membrane containing spin column bycentrifugation at 8000×g for 1 minute. The spin column was washed oncewith a GITC containing washing buffer-like buffer RW1 (QIAGEN GmbH) andtwo times with a ethanol containing buffer-like buffer RPE (QIAGENGmbH).

[0064] A digestion of the residual genomic DNA which could beco-purified with the RNA in low amounts was performed on the silicamembrane according to the instructions in the manual of the RNase-FreeDNase Set (QIAGEN GmbH Cat.No. 79254). The RNA was eluted from thesilica membrane with 2×40 μl of elution buffer. All samples wereprocessed in duplicates. For the analysis, the eluates were diluted1:125 fold and 1 μl of the diluted eluate was analyzed by real timeTaqMan RT-PCR. The mRNA of the GAPDH-gene was amplified using an assaydeveloped by Perkin Elmer. Each sample was analyzed in duplicate in theTaqMan RT-PCR amplification.

[0065] As a control, RNA from the same donor was drawn with a BectonDickinson Vacutainer EDTA tube and was stored in this tube for the sametime period as described above. The RNA from 1 ml of the stored bloodsample was isolated at each time point using TRIzol™LS reagent (LifeTechnologies). The isolated RNA was subsequently cleaned up according tothe RNeasy™ Mini protocol for the RNA clean up (QIAGEN Cat.No. 74103).The RNA was eluted with 2×40 μl of RNase-free water. The eluate wasdiluted 1:50 fold in order to compensate for the lower sample volumeprocessed with the TRIzol method, compared to the 2,5 ml of blood in thesample tubes. The samples were analyzed using also the GAPDH TaqManRT-PCR system from Perkin Elmer.

[0066] The real time RT-PCR results show that in the unpreserved EDTAblood, the transcript level decreases over time (indicated by theincreasing ct value in the TaqMan analysis) up to a degree ofdegradation after 7 to 10 days at which point the mRNA is no longerdetectable. On the other hand, the GAPDH mRNA in the preserved samplesdoes not show any decrease in copy number, taking into considerationthat the error range of the TaqMan assay is ±1 ct value. Within thiserror range, all changes in the ct value have to be considered as normalfluctuations of the amplification system and no degradation is visible.This result clearly indicates the advantage of the new developed bloodcollection device over the EDTA blood collection tube and also makesclear that the stabilization of the RNA is a prerequisite for themolecular analysis of the sample material. It was also possible toisolate the genomic DNA from the stabilized blood sample. Table 3 showsthe results for the stabilization of genomic DNA in human blood. After24 hour and 72 hours of storage at room temperature, isolated genomicDNA was of high molecular weight. The main band migrated at a lengthgreater than 20 kb. The yield was in the range between 47 to 80 μg per2.5 ml of blood, which is within the expected yield range for thisamount of blood. The DNA was also applicable to enzymatic reactions likerestriction endonuclease digestion and PCR amplification. TABLE 3storage at NA stabilization EDTA tube/ mean room temp. device/ct valuemean value/ct ct value value/ct  1 h 33.38 30.17 31.42 29.63 31.48 30.5831.06 32.29 30.06 30.24  1 day 31.28 30.18 28.62 29.35 30.11 31.26 30.3433.2 30.19 32.32  3 days 31.27 33.33 31.92 32.37 30.91 36.32 30.15 4030.3 39.58  7 days 33.03 40 31.16 39.01 32.58 38.4 34.21 37.67 31.936.12 10 days 34.2 40 32.47 40 32.58 38.97 32.36 38.38

EXAMPLE 4 Stabilization of Genomic DNA in Whole Blood

[0067] 2.5 ml blood, drawn with a sodium citrate containing bloodcollection device, were mixed with 6.9 ml of stabilization buffercontaining 4% (w/v) tetradecyltrimethylammonium oxalate and 200 mMtartaric acid in a 16×100 mm polyethylene tube. Samples were stored atroom temperature for 24 hours and 72 hours, respectively. In order toisolate the genomic DNA, the tubes were centrifuged at 5000×g for 10min. The supernatant was discarded and the pellet was washed once withwater. After additional centrifugation at 5000×g for 10 minutes, thepellet was dissolved in 300 μl of a EDTA and sodium chloride containingbuffer and 400 μl of a lysis buffer, i.e., buffer AL (QIAGEN GmbH), and20 μl proteinase K were added. After a proteinase digestion for 10minutes at 65° C., 420 μl of 98% ethanol were added. The sample was thenapplied to a silica membrane containing spin column by centrifugation at8000×g for 1 minute. The spin column was washed once with a guanidinehydrochloride containing washing buffer-like buffer AW1 (QIAGEN GmbH)and once with an ethanol containing buffer-like buffer AW2 (QIAGENGmbH). The DNA was then eluted from the silica membrane with 300 μl of atris-buffer.

[0068] 5 μl of the eluate was analyzed on a 0.8% agarose/TBE gel stainedwith ethidium bromide. The yield of the isolated DNA was determined bymeasuring the optical density at 260 nm wavelength in aspectrophotometer and calculating that 1 OD260 corresponds to aconcentration of 50 μg DNA/ml.

[0069] The genomic DNA was also applied to enzymatic reactions likerestriction enzyme digestion or PCR amplification. For the restrictionendonuclease digestion, 2 μg of the DNA was digested with 6 U EcoRI (E)and Hind III (H), respectively, for 3 hours at 37° C. and analyzedsubsequently on a 0.8 % agarose TBE gel. For the PCR amplification, 150and 300 ng of the DNA were added to a 50 μl total volume PCR reactionmix and a 1.1 kb fragment of the human homologue of giant larvae-genewas amplified. The PCR products were analyzed on a 1.2-% agarose/TBEgel.

[0070] While various embodiments have been chosen to demonstrate theinvention, it will be understood by those skilled in the art thatvarious modifications and additions can be made without departing fromthe scope of the invention as defined in the appended claims.

What is claimed is:
 1. An apparatus for collecting a biological sample,said apparatus comprising: a container defining an internal chamber, anda closure closing said open end, and at least one stabilizing componentcontained within said container in an amount effective to stabilize andpreserve said biological sample.
 2. The apparatus of claim 1, whereinsaid biological sample is whole blood.
 3. The apparaus of claim 1,wherein said stabilizing component is an aqueous solution of astabilizing agent selected from the group consisting of cationiccompounds, detergents, chaotropic salts, ribonuclease inhibitors,chelating agents, and mixtures thereof, and wherein said aqueoussolution of said stabilizing agent has a pH of about pH 2 to about pH12.
 4. The apparatus of claim 3, wherein said aqueous solution has a pHof about pH 2 to about pH
 10. 5. The apparatus of claim 3, wherein saidaqueous solution has a pH of about pH 3 to about pH
 8. 6. The apparatusof claim 1, wherein said stabilizing agent has the general formulaYR₁R₂R₃R₄X wherein Y is nitrogen or phosphorous; R₁, R₂, R₃, and R₄ areindependently selected from the group consisting of branched alkyl,non-branched alkyl, C₆-C₂₀ aryl, and C₆-C₂₆ aralkyl; and X is an anion.7. The apparatus of claim 6, wherein X is an anion selected from thegroup consisting of phosphate, sulfate, formate, acetate, propionate,oxalate, malonate, succinate, citrate, bromide and chloride.
 8. Theapparatus of claim 6, wherein Y is nitrogen and said stabilizing agentis a quaternary amine.
 9. The apparatus of claim 6, wherein said R₁ isan alkyl having 12, 14, or 16 carbon atoms and R₂, R₃, and R₄ aremethyl.
 10. The apparatus of claim 1, wherein said container has aninternal pressure less than atmospheric pressure, for drawing apredetermined volume of said biological sample into said container. 11.The apparatus of claim 3, wherein said stabilizing agent is included inan amount to lyse cells in said biological sample.
 12. The apparatus ofclaim 3, wherein said stabilizing agent lyses reticulocytes, bacteria,red blood cells, white blood cells, and viruses.
 13. The apparatus ofclaim 3, wherein said stabilizing agent is included in an amount topreserve and stabilize nucleic acids in said biological sample.
 14. Theapparatus of claim 3, wherein said stabilizing agent is included in anamount effective to prevent gene induction in said biological sample.15. The apparatus of claim 3, wherein said chaotropic salt is selectedfrom the group consisting of guanidinium isocyanate and guanidiniumhydrochloride.
 16. The apparatus of claim 3, wherein said detergent isselected from the group consisting of sodium dodecylsulfate andpolyoxyethylene sorbitan monolaurate.
 17. The apparatus of claim 3,wherein said ribonuclease inhibitor is placental RNAse inhibitorprotein.
 18. The apparatus of claim 6, further comprising at least oneproton donor in an effective amount to stabilize nucleic acids in saidsample.
 19. The apparatus of claim 18, wherein said portion donor isselected from the group consisting of alkenyl carboxylic acids, C₂-C₆aliphatic mono- and dicarboxylic acids, aliphatic ketodicarboxylicacids, amino acids, mineral acids and mixtures thereof.
 20. A method ofstabilizing a biological sample comprising the steps of: providing asample collection container having a side wall, and a bottom defining aninternal chamber, said container containing at least one stabilizingagent in an amount sufficient to stabilize and preserve a biologicalsample; and obtaining a biological sample and immediately thereafterintroducing said biological sample into said container and mixing saidbiological sample with said stabilizing agent to form a stabilizedbiological sample.
 21. The method of claim 20, wherein said stabilizingagent is in an aqueous medium containing a component selected from thegroup consisting of cationic compounds, detergents, chaotropic salts,ribonuclease inhibitors, chelating agents, and mixtures thereof.
 22. Themethod of claim 20, wherein said container has an internal pressure lessthan atmospheric pressure, said method comprising drawing apredetermined volume of said biological sample directly into saidcontainer and mixing with a stabilizing amount of said stabilizingagent.
 23. The method of claim 22, wherein said biological sample iswhole blood and said method comprises withdrawing said whole bloodsample from a patient and introducing said whole blood sample directlyinto contact with said stabilizing agent in said sample collectioncontainer to lyse cells and stabilize nucleic acids in said whole bloodsample.
 24. The method of claim 20, wherein said biological sample isselected from the group consisting of red blood cell concentrates,platelet concentrates, leukocyte concentrates, plasma, serum, urine,bone marrow aspirates, tissue, and cerebral spinal fluid.
 25. The methodof claim 20, wherein said stabilizing agent is included in an amount toinhibit gene induction in said biological sample.
 26. The method ofclaim 21, wherein said stabilizing agent is a cationic compound havingthe formula the general formula YR₁R₂R₃R₄X wherein Y is nitrogen orphosphorous; R₁, R₂, R₃, and R₄ are independently selected from thegroup consisting of branched alkyl, non-branched alkyl, C₆-C₂₀ aryl, andC₆-C₂₆ aralkyl; and X is an anion.
 27. The method of claim 26, wherein Xis selected from the group consisting of phosphate, sulfate, formate,acetate, propionate, oxalate, malonate, succinate, citrate, bromide andchloride.
 28. The method of claim 26, wherein said R₁ is an alkyl having12, 14, or 16 carbon atoms and R₂, R₃, and R₄ are methyl.
 29. The methodof claim 26, wherein Y is nitrogen.
 30. The method of claim 21, whereinsaid aqueous medium has a pH of about pH 3 to about pH
 8. 31. The methodof claim 21, wherein said chaotropic salt is selected from the groupconsisting of guanidinium isothiocyanate and guanidinium hydrochloride.32. The method of claim 21, wherein said detergent is selected from thegroup consisting of sodium dodecylsulfate and polyoxyethylene sorbitanmonolaurate.
 33. The method of claim 21, wherein said ribonucleaseinhibitor is placental RNAse inhibitor protein.
 34. The container ofclaim 26, further comprising at least one proton donor in an effectiveamount to stabilize nucleic acids in said sample.
 35. The container ofclaim 34, wherein said proton donor is selected from the groupconsisting of alkenyl carboxylic acids, C₂-C₆ aliphatic mono- anddicarboxylic acids, aliphatic ketodicarboxylic acids, amino acids,mineral acids and mixtures thereof.
 36. A method of collecting andstabilizing a whole blood sample, said method comprising: providing asample collection container having a side wall, a bottom wall and aclosure member forming an internal chamber, said container enclosing aneffective amount of an aqueous solution or dispersion of a nucleic acidstabilizing agent to stabilize and preserve nucleic acids in a wholeblood sample, said internal chamber having pressure less thanatmospheric pressure; and collecting a whole blood sample directly froma patient in said collection container and mixing said blood sample withsaid stabilizing agent to form a stable whole blood sample.
 37. Themethod of claim 36, wherein said closure is a septum and said methodcomprises piercing said septum with a cannula and introducing said wholeblood sample through said cannula into said collection container. 38.The method of claim 36, wherein said stabilizing agent is selected fromthe group consisting of cationic compounds, detergents, chaotropicsalts, ribonuclease inhibitors, chelating agents, and mixtures thereof,and wherein said aqueous solution or dispersion has a pH of about pH 2to about pH
 12. 39. The method of claim 36, wherein said aqueoussolution or dispersion has a pH of about pH 2 to about pH
 5. 40. Themethod of claim 36, wherein said aqueous solution or dispersion has a pHof about 3.6 to about pH 3.8.
 41. The method of claim 36, wherein saidstabilizing agent is selected from the group consisting of guanidiniumisothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate,polyoxyethylene sorbitan monolaurate, placental RNAse inhibitor protein,and mixtures thereof.
 42. The method of claim 38, wherein saidstabilizing agent has the general formula YR₁R₂R₃R₄X wherein Y isnitrogen or phosphorous R₁, R₂, R₃, and R₄ are independently selectedfrom the group consisting of branched alkyl, non-branched alkyl, C₆-C₂₀aryl, and C₆-C₂₆ aralkyl; and X is an anion.
 43. The container of claim18, wherein said portion donor is selected from the group consisting ofalkenyl carboxylic acids, C₂-C₆ aliphatic mono- and dicarboxylic acids,aliphatic ketodicarboxylic acids, amino acids, mineral acids andmixtures thereof.